Not applicable.
The present invention is generally related to inhibiting the growth or proliferation of cancerous cells. In particular, the invention is directed to novel methods for assaying for modulators of GADD45 polypeptide activity. The invention also provides means to sensitize a proliferating cell to a DNA base-damaging agent by administration of novel inhibitors of GADD45 polypeptide activity, and compositions which modulate GADD45 polypeptide activity.
A common method to treat cancer is to give radiation or chemicals to damage the cancer cell""s chromosomes (DNA) so badly that the cell dies. These treatments are, however, equally toxic to cells growing normally. It is difficult or impossible under most circumstances to limit a patient""s exposure to the DNA damaging agent to only the cancer cells. One way to protect normally growing cells from the toxic anti-cancer treatment would be to simultaneously treat the cancer cells with a second agent whose activity reduces the amount of toxic radiation or chemical needed to be effective. This could be done by xe2x80x9csensitizingxe2x80x9d the cancer to the toxic treatments so the growing tumor cells die when a smaller amount of toxic radiation or chemical is administered.
One way to xe2x80x9csensitizexe2x80x9d an undesirable growing cell (such as a cancer cell) to a DNA-damaging agent is to manipulate the cell so it will die when it incurs less DNA damage. A proliferating cell can detect if and how much chromosome damage it has. If there is enough DNA damage, a normal cell commits suicide, killing itself by a preprogrammed cell death mechanism called xe2x80x9capoptosis.xe2x80x9d This suicidal reaction is a protective mechanism because damaged DNA can result in mutation of genes and disease, such as cancer (Hartwell (1994) Science 266:1821-1828).
A damaged cell, however, actively tries to avoid suicide by repairing the chromosomal damage inflicted by the toxic treatments. By using its DNA repair machinery, the cell can save itself from what otherwise would be a suicide-initiating dose of radiation or base-damaging agent (Smith (1996) Mutation Research 340:109-124).
One avenue by which a cell can repair enough DNA damage to xe2x80x9csave itselfxe2x80x9d from a suicidal fate takes advantage of the fact that the programmed cell death signal is triggered only at the stage of the cell""s growth where its chromosomes begin to divide (called xe2x80x9cmitosisxe2x80x9d). If the cell has the time to repair enough damaged DNA before entry into mitosis it can avoid suicidal apoptosis. To gain sufficient repair time after detecting that its chromosomes have been damaged, the cell xe2x80x9cstallsxe2x80x9d its cell cycle at a stage just before entry into mitosis. If the damage is too great, not enough repair can be done and the cell dies (Paulovich (1997) Cell 88:315-321; O""Connor (1997) Cancer Surveys 29:151-182). Under xe2x80x9cnormalxe2x80x9d circumstances, with an intact DNA repair and cell cycle xe2x80x9cstallingxe2x80x9d mechanisms, relatively larger of amounts of toxic agent must be administered to kill the growing cell. However, if this stalling mechanism can be disturbed, less DNA repair time is available, less DNA damage is fixed before entry into mitosis, and thus relatively lesser amounts of anti-cancer agent are needed to kill the cell. Thus, discovery of inhibitors of this stalling mechanism, and their co-administration with DNA base-damaging agents, would provide novel means of effectively treating cancer with lower doses of toxic agents.
G2/M checkpoints prevent the segregation of damaged chromosomes, which is likely to be important in human tumorigenesis. (Hartwell, L. H. and M. B. Kastan, Science 266:1821-1828 (1994); Paulovich, A. G., et al. Cell 88:315-321 (1997).) The transition from G2 to M is regulated, in part, by the G2-specific kinase consisting of Cdc2 and cyclin B1. (Nurse, P., Cell 79:547-550 (1994).) Many G2/M regulatory genes have been identified recently, such as Chk1, Chk2, ATM (MEC1 and TEL1 in S. cerevisiae and RAD3 in S. pombe) and the 14-3-3 family. (Agarwal, M. L., et al., Proc. Natl. Acad. Sci. USA 92:8493-8497 (1995); Elledge, S. J., Science 274:1664-1672 (1996); Morrow, D. M., et al., Cell 82:831-840 (1995); Paulovich, A. G., et al. supra; Savitsky, K., et al., Science 268:1749-1753 (1995); Weinert, T. A., et al., Genes Dev. 8:652-665 (1994).) Their products alter Cdc2 activity by inhibiting dephosphorylation of inhibitory sites on Cdc25C. p53 also has been implicated in an IR-induced G2/M checkpoint. (Agarwal, M. L., et al., supra; Bunz, F., A., et al., Science 282:1497-1501 (1998); Guillouf, C., et al., Oncogene 10:2263-2270 (1995); Powell, S. N., et al., Cancer Res 55:1643-1648 (1995); Stewart, N., et al., Oncogene 10:109-115 (1995).) It may modulate the G2/M transition by upregulating 14-3-3"sgr" (12) and/or p21waf1. (Bunz, F., A., et al., Science 282:1497-1501 (1998); Dulic, V., et al., Mol. Cell Biol 18:546-557 (1998); Medema, R. H., et al., 16:431-441 (1998).) Consequently, cells lacking p53 show chromosome instability (Fukasawa, K., et al., Science 271:1744-1747 (1996)), a phenotype likely resulting from defects in the G2/M checkpoint. Therefore, a multiplicity of G2/M checkpoints in response to DNA damage may well involve redundant controls involving both p53-independent and p53-dependent pathways.
GADD45 is a 165-amino acid nuclear protein whose expression also is p53-dependent (Zhan, Q., et al., Mol. Cell Biol 13:4242-4250 (1993)). GADD45 was originally identified on the basis of a rapid induction in Chinese hamster ovarian cells after UV irradiation (Fornace, A. J., Jr., et al. Mol. Cell Biol 9:4196-4203 (1989).). Induction of GADD45 also was observed following treatment with many other types of DNA-damaging agents, including various environmental stresses, hypoxia, IR, genotoxic drugs and growth factor withdrawal. (Papathanasiou, M. A., et al., Mol. Cell Biol 11:1009-1016 (1991)). In mammalian cells, two additional family members with extensive sequence homology, GADD45xcex2 and GADD45xcex3, were identified recently. (Takekawa, M. and H. Saito, Cell 95:521-530 (1998).) Similar to p53-deficient cells, cells from Gadd45-deficient mice also show genomic instability, including chromosome abnormalities and centrosome amplification. (Hollander, M. C., et al., Nat. Genet 23:176-184 (1999).) It is known that GADD45 binds to PCNA, p21waf1 and Cdc2. (Kearsey, J. M., et al., Oncogene 11:1675-1683 (1995); Smith, M. L., et al., Science 266:1376-1380 (1994); Zhan, Q., et al., Oncogene 18:2892-2900 (1999).) GADD45 has no inhibitory effect on the kinase activity of the G1-specific Cdk2/cyclin E complex. (Smith, M. L., et al., Science 266:1376-1380 (1994); Zhan, Q., et al., Oncogene 18:2892-2900 (1999).) Increased expression of GADD45 in primary human fibroblasts arrests cells at the G2-M boundary. This arrest was attenuated by the overexpression of cyclin B1 and Cdc25C.
Thus, GADD45 is a mediator of the G2-M stalling mechanism. It has been reported that blocking GADD45 expression by constitutive antisense oligonucleotide expression xe2x80x9csensitizedxe2x80x9d a human colon carcinoma cell line to killing by UV irradiation and by cisplatin, a DNA-damaging cancer chemotherapy drug (Smith (1996) Oncogene 13:2255-2263). Identification of novel modulators, particularly inhibitors, of GADD45 polypeptide activity would provide new means to inhibit proliferation of cancer cells. The invention fills these, and other, needs.
The invention provides various in vitro methods of assaying for modulators of GADD45 polypeptide activity. In one embodiment, the method comprises combining a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease GADD45 polypeptide binding specifically to a Cdc2 polypeptide. In another in vitro embodiment, the method comprises combining a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease GADD45 polypeptide-mediated dissociation of a Cdc2/Cyclin B1 protein complex. In one embodiment, the method comprises combining a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease the ability of GADD45 polypeptide to bind specifically to chromatin. In another embodiment, the method comprises combining a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease GADD45 polypeptide inhibition of histone phosphorylation by a Cdc2/Cyclin B1 complex. In alternative embodiments, the GADD45 polypeptide is a subsequence of the full length protein (SEQ ID NO:2) that includes all or part of an required for GADD45 activity, for an active site or for a protein:protein binding domain, such as, e.g., a peptide having a sequence as set forth by amino acid residues 71 to 124, or 61 to 87, or 71 to 87, or 61 to 124, of SEQ ID NO:2.
The invention also provides an in vitro method of assaying for a compound capable of specifically binding to a GADD45 polypeptide, wherein the method comprises combining a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound specifically binds to the GADD45 polypeptide. In alternative embodiments, the GADD45 polypeptide is a subsequence of the full length protein that includes all or part of an active site or protein:protein binding domain, such as, e.g., a peptide having a sequence as set forth by amino acid residues 71 to 124, or 61 to 87, or 71 to 87, or 61 to 124, of SEQ ID NO:2.
The invention also provides in vivo methods of assaying for a modulator of GADD45 polypeptide activity. In one embodiment, the method comprises combining a cell expressing a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease GADD45 polypeptide binding specifically to a Cdc2 polypeptide. In another embodiment, the method comprises combining a cell expressing a GADD45 polypeptide with a test compound in an aqueous solution and assaying whether the test compound can inhibit or decrease GADD45 polypeptide-mediated dissociation of a Cdc2/Cyclin B1 protein complex. The cells can be proliferating cells, such as cancer cells.
The invention provides methods for sensitizing a proliferating cell to a DNA base-damaging agent by inhibiting GADD45 polypeptide activity. In one embodiment, the method comprises administering a composition capable of specifically binding to a GADD45 polypeptide in an amount sufficient to inhibit or decrease GADD45 polypeptide specific binding to the Cdc2 polypeptide. Another embodiment comprises administering a composition capable of specifically binding to the GADD45 polypeptide in an amount sufficient to inhibit or decrease GADD45-mediated dissociation of a Cdc2/Cyclin B1 protein complex. In these methods, the composition can be an antibody specifically reactive with the GADD45 polypeptide. In one embodiment, the antibody is specifically reactive with a GADD45 active site or protein:protein binding domain. In alternative embodiments, the GADD45 is a subsequence of the full length protein that includes all or part of an active site or protein:protein binding domain or a site required for GADD45 polypeptide activity, such as, e.g., a peptide having a sequence as set forth by amino acid residues 71 to 124, or 61 to 87, or 71 to 87, or 61 to 124, of SEQ ID NO:2. In various embodiments, the proliferating cell can be a cancer cell; the DNA base-damaging agent can be UV radiation or a DNA base-damaging agent such as a chemotherapeutic agent, and the chemotherapeutic agent can be a base-damaging alkylating agent.
The invention also provides inhibitors of GADD45 polypeptide activity in the form of GADD45 peptides comprising sequences which are required for GADD45 activity. These GADD45 peptides, when administered to a cell, interfere with the ability of GADD45 to inhibit a Cdc2/cyclin B1 protein complex from phosphorylating histone H1. In alternative embodiments, the blocking GADD45 peptides comprise amino acids 62-67 of SEQ ID NO:2, and can, for example, have a sequence as set forth by amino acid residues 61 to 87 of SEQ ID NO:2. Alternatively, they can be other subsequences of the GADD45 protein which comprise the DEDDDR (SEQ ID NO:5) acidic motif (amino acid residues 62-67 of GADD45) and a portion of the native sequence of SEQ ID NO:2 flanking that motif on the amino or the carboxyl sides, or both. In various embodiments, the GADD45 peptides can be 10, 20, 30, 40, 50 or 60 amino acid residues in length. These peptides, like the GADD45 binding inhibitors of the invention, can be co-administered with cancer therapeutic agents, particularly those which damage DNA (as, e.g., UV irradiation or chemotherapeutic agents such as cisplatin) to sensitize proliferating cells to the DNA damaging anti-cancer agent. The invention further provides polypeptides which interfere with or inhibit the ability of GADD45 to reduce or eliminate phosphorylation of histone H1. Typically, such polypeptides also comprise the acidic motif DEDDDR (SEQ ID NO:5).
The polypeptides of the invention can be in a pharmaceutically acceptable carrier, and can be loaded into liposomes or otherwise protected from exposure to proteases before they reach an intended site of action. The invention also provides nucleic acids encoding the polypeptides and for the administration of the polypeptides or the nucleic acids encoding them to inhibit the growth or proliferation of cancer cells by rendering them more sensitive to chemotherapeutic agents or to radiation therapy.